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The overall goal of this procedure is to simultaneously quantify multiple proteins on the chip using sandwich immunoassays in a spotter-free manner without cross reactivity between reagents. This method can help answer key questions in the quantitative proteomics field, such as the discovery and validation of candidate protein biomarkers in cancer. The main advantages of this technique are that it has cross reactivity between antibodies and antigen in multiplexed sandwich assays and that and users do not need a complex microarray spotter.

Demonstrating the procedure will be Heidi Larkin, a scientific director at Parallex BioAssays. To begin preparing slides for the single-transfer method, fix a new transfer slide on the ink jet microarray spotter deck. Using a polystyrene microbead solution, or 30%glycerol, spot an array of alignment marks on the edge of the slide.

Using the instrument software and spotter camera, take a picture of the alignment marks for quality control. Next fix a nitrocellulose or functionalized glass assay slide to the left of the transfer slide on an ink jet micro array spotter deck. Using alignment marks, spot 24 arrays of capture antibody solutions with 400 micrometer center-to-center spacing.

Incubate the assay slide at room temperature in an atmosphere with 60%relative humidity for one hour. Then clip a 24-well silicone gasket onto the slide, aligned with the arrays. Pipette 80 microliters of a 0.1%solution of polysorbate 20 in PBS into each well.

Shake the slide at 450 rpm for five minutes and discard the solution. Wash the slide in this way three times. Next load 80 microliters of BSA free-blocking solution into each rinsed well.

Shake the slide at 450 rpm for one hour. Then discard the solution, remove the silicone gasket, and dry the assay slide using either nitrogen gas or a centrifuge. Seal the assay slide in an airtight bag with silica gel or another desiccant and store it at minus 20 degrees Celsius.

Using the same alignment marks, spot 24 arrays of detection antibody solutions using 3.2 nanoliters per spot in a center-to-center spacing of 400 micrometers. Store the transfer slide at minus 20 degrees Celsius in a sealed bag with a desiccant. To begin preparing slides for the double-transfer method, fix a transfer slide on an ink jet microarray spotter deck.

Spot 24 arrays of capture antibody solutions with the center-to-center spacing of 400 micrometers. Then turn the snap apparatus plunger switches 90 degrees to lock the plungers in the open position. Fit the capture transfer slide into the apparatus with the clipped corner against the fixed pogo pin.

Release the pogo pins by turning the corresponding plunger switches back by 90 degrees. Ensure that the transfer slide is firmly held by the pogo pins and is flush with the fixed alignment pins. Then place a new nitrocellulose or a functionalized glass slide on the pogo pins with the coated side face down.

Turn the other three switches to release the straight pins. Verify that both slides are held in place against the fixed alignment pins. Then fit the top shell onto the slide holder with the pillar seated in the alignment holes.

Insert the closed apparatus into the snap cage and fully depress the closure tab. Leave the apparatus closed for one minute. Then pull up the closure tab to release the apparatus from the cage.

Remove the top shell and separate the assay and transfer slides. Incubate the assay slide for one hour. Next clip a 24-well silicone gasket onto the slide and wash the slide three times.

Then shake the slide with BSA-free blocking solution for one hour. Remove the gasket and dry the assay slide under a stream of nitrogen gas or a centrifuge. Next fix another transfer slide on the ink jet spotter deck.

Spot 24 arrays of detection antibody solutions on the slide with 3.2 nanoliters per spot in a center-to-center spacing of 400 micrometers to make the transfer slide. Store the detection transfer slide at minus 20 degrees Celsius. To begin the immunoassay remove the prepared assay slide from the freezer and allow it to warm to room temperature in its sealed bag for 30 minutes.

Prepare seven serially diluted solutions of proteins in PBS with 0.05%polysorbate 20. Prepare appropriately diluted sample solutions in the same buffer. Clamp a 24-well silicone gasket onto the room temperature assay slide.

Load the seven protein dilutions into seven of the eight wells in a column. Load protein-free PBS with 0.05%polysorbate 20 into the last well of that column. Load the sample solutions into the remaining wells.

Shake the slides at 450 rpm for one hour at room temperature or at 4 degrees Celsius overnight. Then wash the slide three times. Remove the gasket and dry the slide under a stream of nitrogen gas.

Next allow the detection antibody transfer slide to warm to room temperature for 30 minutes in its sealed bag. Then incubate the slides for 20 minutes in a closed chamber with the relative humidity of 60%to rehydrate the arrays. Use the pogo pins to fix the detection transfer slide face up in the snap apparatus.

Place the assay slide face down on the pogo pins and align the slide with the straight pins. Close the apparatus and snap transfer the detection antibodies to the assay slide. Remove the slides from the apparatus and incubate the assay slide for one hour.

Clamp a 24-well silicone gasket to the slide and wash the slide three times. Then load into each well 80 microliters of a 2.5 microgram per milliliter solution of streptavidin fluoro-4 in PBS. Shake the slide at 450 rpm for 20 minutes.

Wash the slide three times with a 0.1%solution of polysorbate 20 in PBS and once with distilled water. Then remove the gasket and dry the slide. Scan the slide with a fluorescent scanner and measure the spot intensities.

Determine the limits of detection in the protein concentrations. Two fluorescent proteins were sequentially patterned on a nitrocellulose-coated slide using the double-transfer method with a 16-wells template. No cross-contamination was observed upon transferring the second fluorescent protein.

Smaller spots with decreased spot-to-spot distance could be transferred to a glass slide with a highly hydrophobic surface. The double-transfer method resulted in minimal angular misalignment of the arrays. The double-transfer method was used to perform a 50-plex immunoassay of breast cancer-related proteins in the sandwich assay format.

No cross reactivity was observed, allowing independent optimization of each antibody pair. Standard curves calculated from the assays indicated that 35 of the 50 proteins had picogram per milliliter limits of detection. Once mastered this technique can be completed in approximately three hours.

This technique allows scientists to conduct microarray-based assay and to deliver reagents as nano droplets. While attempting this procedure, remember to fix the slides properly on the spotting deck and in the snap apparatus. After watching this video, you should have a good understanding of how to use the snap apparatus for cross-reactivity-free multiplexed sandwich immunoassays.